Scaling Down The Thermocyclers

The PCR industry is shifting its focus away from macro-scale systems and is focused on developing the micro-scale devices.

The Nobel Prize winning polymerase chain reaction (PCR) technology has revolutionized molecular biology since its invention in 1986 and is doing the same for medical diagnostics for over the last 15 years. Thermal cyclers or PCR machines are being used in laboratories to facilitate other temperature-sensitive reactions, including, but not limited to, restriction enzyme digestion or rapid diagnostics. They are now gaining an increasing role in clinical diagnosis of infectious disease, nucleic acid amplification, paternity testing, DNA fingerprinting, detection and diagnosis of infectious disease, quality control, and personalized medicine owing to continuous technological advancements. 

In the early development of thermal cyclers, the cooling system relied on a bulky plumbing compressor, which made it impossible to have a small-footprint instrument. In the present day, solid-state Peltier blocks are utilized in thermal cyclers enabling the modern machines to complete more PCR runs in a day. Advances are also under-research for replacement of Peltier blocks with more efficient thermoelectric devices to further increase PCR runs. Also, a heated lid is now a common feature of thermal cyclers to prevent evaporation and condensation of PCR samples during runs. Likewise, many of today’s thermal cyclers are built with flexibility in sample throughput in mind. With interchangeable blocks, a benchtop thermal cycler may accommodate, for example, from one to 480,000 amplification reactions. For high-throughput automation, thermal cyclers designed for hands-free operations and integration with robotic liquid-handling platforms are now available.

Besides the block technology, algorithms to control sample temperatures have also improved over the years. Complex mathematical models are applied for more precise regulation of block temperatures to achieve uniform heating and cooling of the PCR samples. Thermal cyclers today are designed for easy programming of PCR protocols and are equipped with intuitive user interfaces, such as touch screens and easy programming features, enabling faster and more efficient protocol setup. Recent advances also allow convenient access to thermal cyclers anytime and from anywhere, using a mobile device or desktop computer. Connectivity to the cloud offers enhanced accessibility at fingertips and freedom to create and share protocols as well as to schedule, start/stop, and monitor PCR runs.

Technological advances
PCR technology has expanded and changed tremendously since its introduction. The initial method was extremely labor-intensive and time-consuming, but important technological improvements and novel variations in instruments have simplified the process and made clinical implementation much easier.

Changes in Peltier blocks. Using thicker metal blocks, including those made of silver, which is highly heat conductive, improves the conductivity of the heat exchange block. However, the increased thermal mass increases the time taken to raise the entire blocks to the same even temperature, and can lead to undershoots and overshoots of temperature. In a new approach, the use of a hollow heat exchange block with a circulating conductive fluid improves temperature control and heat uniformity, for example, the current real-time PCR systems available in the market that take around 40 minutes for 40 cycles. Another step away from Peltier-based technology involves the use of a ceramic heating plate in its cycler, heating samples in disposable tubes, and then cooling using force-air cooling. These systems take 20–40 minutes for 40 cycles.

qPCR leading the way. In recent decades, the advert of the real-time quantitative PCR (qPCR) is gaining popularity for the detection of pathogens in clinical microbiology. The majority of diagnostics is and will be based on real-time or qPCR technology that requires less than five hours for detection of pathogens and is simple, reproducible, and has improved quantitative capacity over conventional PCR. Recognizing this importance of molecular diagnostics for early diagnosis in life threatening infections, many manufacturers have recently launched qPCRs for invasive disease. Now, the qPCR has also been adapted to detect RNA viruses such as HIV and hepatitis C and the analysis of RNA transcripts associated with some cancers. A new ultra-rapid technology in development could promise a more efficient process, particularly in analyzing large numbers of samples. The technology utilizes a genetically engineered thermostable reverse-transcriptase so the PCR process can proceed directly from RNA.

Miniaturizing thermal cyclers. PCR is one of the most important (research) technologies, and yet it is one of the most limiting when one is outside of the lab because of the size and cost of the instrument. Most thermal cyclers control their temperatures using Peltier junctions, thermoelectric devices that can switch rapidly between heating and cooling. Unfortunately, Peltier junctions are inefficient, and the components required to operate them keep PCR machines heavy and greedy for electricity. However, this can be tackled by the use of a novel approach, wherein samples are heated with a thin-film resistive heater similar to the window defrosters found in cars and are cooled using a simple fan. A microcontroller drives the heating, cooling, and incubation cycles. This simpler design makes the machine much smaller and lighter than ordinary thermal cyclers, and has other benefits too, such as low cost.

Improving instrumentation. Initial commercial instruments required laser light sources and/or heat-block elements that were relatively slow to cycle to the required temperatures, or utilized thin capillaries that were easily broken and hard to use. Significant technological improvements include  the utilization of LED light sources, redesign of the heat-block elements to improve cycling times, additional fluorescent detectors, and the identification of alternative strategies to generate fluorescent signals. These enhancements have made it possible to perform a PCR assay in the traditional 96-well PCR plate in as little as 20–30 minutes.

Outlook
Thermal cyclers have evolved in technology and design since their introduction and innovations continue to facilitate improvement in PCR and advances in molecular biology research. However, the commercial thermal cyclers are still bulky, expensive, and limited for laboratory use only. As such, it is difficult for on-site molecular screening and diagnostics. The industry is thus shifting its focus away from macro-scale systems and is focused on developing the micro-scale devices that offer various benefits such as less sample requirement, less reaction time, and also offers cost-effective benefits, as many microfluidic devices are manufactured from inexpensive polymers.

The potential for PCR diagnostics in the future is huge. It would be easier to define where PCR machines will not be used – everything in this planet has a DNA or RNA genome so if one wants to detect anything they go to PCR. Now marking its 35th anniversary, the PCR machine has become a ubiquitous laboratory tool. Nonetheless, researchers, engineers, and physicians are still finding ways to propel it into new territories. A sampling of a few of these efforts shows just how far PCR’s reach has grown – from dairies to clinics, and from classrooms to outer space. Whether manufacturers are making portable thermal cyclers, using cutting-edge PCR tests for rapid cancer diagnosis, or exploring other techniques to break free of PCR’s limitations, experts in the field are optimistic about the next 35 years of DNA amplification. It is an exciting time!